L-tryptophan, a kind of essential amino acid, has been widely used as a feed and food additive. L-tryptophan is generally produced by fermentation using microorganisms of the genus Corynebacterium, Bacillus or Escherichia, and variant thereof. L-tryptophan is biosynthesized from chorismic acid which is a common intermediate for the synthesis of aromatic amino acids. Specifically, L-tryptophan is biosynthesized by the action of five enzymes, which are synthesized by the tryptophan operon trpEDCBA, on chorismic acid produced through the common shikimic acid pathway that starts from phosphoenolpyruvate and D-erythrose-4-phosphate. First, anthranilate synthase (TrpE-TrpD complex) acts on chorismic acid to synthesize anthranilic acid, then anthranilate phosphoribosyltransferase (TrpD) acts on it to synthesize N-(5′-phosphoribosyl)-anthranilate. Then, phosphoribosylanthranilate isomerase (TrpC) and indole-3-glycerol phosphate synthase (TrpC) act on the N-(5′-phosphoribosyl)-anthranilate to synthesize indole-3-glycerol-phosphate, then tryptophan synthase (TrpB-TrpA complex) acts on it to synthesize L-tryptophan (FIG. 1; Bonggaerts et al., Metab Eng, 3, 289-300, 2001).
In E. coli, a protein encoded by a complex of the trpE and trpD genes forms anthranilate synthase to synthesize anthranilic acid. The synthesized anthranilic acid reacts with 5-phosphoribosyl 1-pyrophosphate (PRPP) by anthranilate phosphoribosyltransferase encoded by the trpD gene, thereby synthesizing N-(5′-phosphoribosyl)-anthranilate.
Herein, the protein encoded by the trpD gene acts as anthranilate synthase in combination with the protein encoded by the trpE gene or acts alone as anthranilate phosphoribosyltransferase, and a wild-type E. coli strain maintains the balance of such two actions.
In a wild-type E. coli strain, the concentration of PRPP that is used as a cofactor in intracellular tryptophan biosynthesis is about 180 μM (Bennett et al., Nat Chem Biol, 5, 595-599, 2009). In an L-tryptophan-producing strain with enhanced biosynthesis of anthranilic acid, the extracellularly accumulated concentration of anthranilic acid is at the maximum value, whereas the intracellular concentration of PRPP is greatly lower than the concentration of anthranilic acid. Low PRPP concentration acts as a limiting factor in the production of N-(5′-phosphoribosyl)-anthranilate to reduce the overall synthesis rate of L-tryptophan and increase the intracellular and extracellular accumulation of anthranilic acid. Thus, the use of an anthranilate phosphoribosyltransferase having higher affinity for PRPP makes it possible to prevent the intracellular and extracellular accumulation of anthranilic acid and to increase the biosynthesis rate of L-tryptophan.
Among previous reports on improvement in L-tryptophan-producing strains, a technology of enhancing the biosynthesis pathway while maintaining the balance of anthranilate synthase and anthranilate phosphoribosyltransferase activities has not yet been reported.
According to studies, the Km value of E. coli for PRPP is 50, whereas the Km value of yeast for PRPP is about 22.4±2.6 (Hommel et al., Eur J Biochem, 180, 33-40, 1989). Thus, the affinity of yeast anthranilate phosphoribosyltransferase for PRPP is higher than that of E. coli anthranilate phosphoribosyltransferase. Thus, it is expected that the use of yeast anthranilate phosphoribosyltransferase will have greater effects on the prevention of the intracellular and extracellular accumulation of anthranilic acid and an increase in the biosynthesis of L-tryptophan in microorganisms with enhanced L-tryptophan biosynthesis pathways.
During an attempt to enhance the chorismic acid biosynthesis pathway and trp operon pathway in E. coli in order to produce a large amount of industrially useful L-tryptophan, the present inventors have found that, as the above two pathways are enhanced, anthranilic acid is accumulated intracellularly and extracellularly, and for this reason, abnormal culture appears. Accordingly, the present inventors have continued to study under the expectation that enhancement of the expression of anthranilate phosphoribosyltransferase in microorganisms having an enhanced L-tryptophan biosynthesis pathway can prevent the intracellular and extracellular accumulation of anthranilic acid and increase the biosynthesis of L-tryptophan, thereby completing the present invention.